1. Field of the Invention
The present invention relates to the field of biology, and more particularly, to a two-stage nucleic acid reaction and detection tube which is to perform polymerase chain reaction (PCR) and/or nucleic acid detection and can be directly processed in the same tube without any liquid transfer.
2. Description of the Prior Art
Polymerase chain reaction (hereinafter referred to as “PCR”) is a technology for rapid amplification of DNA, and its principle and the main operational steps may include: (a) denature: using a relatively high temperature (90˜95° C.) for the double-stranded DNA dissociating into single stranded DNA, which is then used as a template for replication; (b) primer annealing: when the temperature is lowered to a predetermined temperature, primers will be adhered to the correct positions of the target genes; (c) primer extension: the reaction temperature will be adjusted to 72° C., in combination of using magnesium ions as enzyme cofactors, the deoxy-ribonucleotide triphosphate (hereinafter referred to as “dNTPs”) will be sequentially adhered to and after the primer by DNA polymerases, in accordance with the base sequence of nucleotides on the template, so as to form a synthetic DNA fragment. By repeating the three-step process of temperature oscillation, the number of the target gene can be doubled in each repetition of the three-step process, thus can be 109 times after 40 cycles of the three-step process. The signal of the target gene can therefore be amplified. Accordingly, the PCR detection technology is generally used for detecting molecular signals in clinical diagnosis, such as pathogen diagnosis, diagnosis of genetic disease, diagnosis of cancer tumors, or the like. The RT-PCR technique which is derived from PCR also has similar principle and application, therefore is widely used in current techniques clinical diagnosis.
Devices used to perform PCR or RT-PCR reactions often include heat resistant plastics as the materials of the reaction tube. The amplification of nucleic acid is achieved by using the thermostat metal to repeatedly increase and decrease the temperature for the tube so as to reach different temperatures in each three-step process. In current system, the system with thermostat metal requires a relatively lager space, the entire temperature control system may occupy a larger space and heat capacity ratio. In addition, according to current practice of operation, one test required 30-35 cycles and the time required for the reaction is about two to three hours, consequently, most of the time of the process relies on waiting for the rise of the temperature or cooling metal, making it difficult to reduce the reaction time.
In addition, the amplification of the target gene in PCR or RT-PCR is often conformed by gel electrophoresis, which can separate the nucleic acid by its molecular size. After the PCR or PT-PCR process, the product containing amplified target gene (hereinafter “the product”) is transferred from the tube into a previously prepared gel well and since the nucleic acid contains negatively charge in neutral or alkaline solution, it will move toward the anode wherein the moving speed is proportional to it molecular weight. By this process, it can be checked if the amplification of nucleic acid successes or not. However, even the gel electrophoresis detection exhibits high accuracy, it still takes several hours to complete the PCR/RT-PCR and gel electrophoresis. For those diagnoses which are willing to obtain results in short time, it would be not suitable to use PCR/PT-PCR to approach the diagnosis or inspection. In addition, when performing the gel electrophoresis, the product of PCR/RT-PCR needs to be transferred from the reaction vessel to the gel well, and the product is easily contaminated during transferring, which may result in false positive.
To shorten the reaction time, a technology that uses thermal convection for performing PCR has been developed (hereinafter “thermal convection PCR”). This technology was first designed by Krishnan et al., T and it uses a cylindrical tube of the Rayleigh-Benard cell with two different heating sources which are disposed at two corresponding sites of the tube. In general, the top level of the reagent is maintained at around a temperature of about 60° C., while the bottom temperature is about 95° C. By the temperature differences arising from the cylindrical cavity through the upper and lower end, it can drive the flow of fluid in the chamber, thus processing the PCR reaction. This embodiment may also be applied to RT-PCR. From this, other technology derived from it with similar principle has been developed for commercial use, such as the use of isolated single point of heating technology called “insulated isothermal polymerase chain reaction” (iiPCR), which performs the RCR reaction in a closed capillary in combination of three points heating sources; or by using non-contact irradiation heating source, which contains heating point of the cylindrical tube closed loop design to achieve the effect for the PCR or RT-PCR. By using the thermal convection process, PCR as well as RT-PCR will not need to use thermostat metal to be repeated temperature oscillation of three steps of the reaction temperature, so it can save a lot of repeated heating and cooling time, thus reducing the use of temperature control metal.
In order to avoid the product of the PCR/RT-PCR from being contamination, which would result in false positive, there are more and more detection technologies used to replace original gel electrophoresis, such as using a specific binding fluorescence chemical compound to combine the target gene. When a laser beam with appropriate wavelength is applied, the chemical compound will emit fluorescence, and it will be detected by the equipment. The intensity of the fluorescence is proportional to the amount of the product of PCR/RT-PCR, so the method can reach in-situ qualitative or even quantitative inspection, thus reducing the reaction temperature.
In another aspect, there is one common inspection technology that uses a specific binding dipstick for the target gene to detect the product of PCR/RT-PCR. The method includes using two different antigens that can specifically bind antibody, for example, the first type of antigen is DIG or TexasR, and the second type of antigen is Avidin or FITC. After the reaction, the product would contain these two types of antigens. In addition, one side of the dipstick is attached to an Ag gel, emulsion beads or other coloring compounds, which can specifically bind the antibodies of the above-mentioned antigens, such as Biotin-anti-FITC or other specific binding protein; the other side of the dipstick is attached to a cotton pad; and some portions of the dipstick is coated to the antibody of the first type of antigens such as anti-DIG, anti-TaxasR or other specific binding antibody. After the reaction, the product of PCR or RT-PCR is transferred to the end of the lipstick that contains the coloring compound, and the first type of antigen will specifically bind the first type of antibody to forma first type antigen-antibody-coloring complex. The complex will then move toward the terminal end with cotton pad. When it moves at the part containing the second type of antibody, the second type of antigen will specifically bind the second type of antibody and thus exhibit colors. The user can therefore obtain the result according to the colors. Since there are only several minutes that are used to complete the dipstick detection, it can save more time in comparison to convention electrophoresis method. In other situation, the antibody can be replaced by the nucleic acid probe depending on different requirement.
As described above, since the thermal convection PCR and the dipstick detection method can save a lot of time comparing to convention PCR/RT-PCR and gel electrophoresis method, it is widely used in the industry in this field. However, there is still not found a detection tube that can operate both thermal convection PCR and the dipstick detection method in single tube. Conventional method is to complete the thermal convection PCR in one tube and then to transfer the reagent onto the dipstick so as to carry out the entire detection method. However, since the product is difficult to take out from the tube, it is not such convenient to operate the system, and it also raises the risk of contamination.
Accordantly, there is still a need to have a two-stage detection tube that can both perform polymerase chain reaction (PCR) and/or nucleic acid dipstick detection and can be directly processed in the same tube without any liquid transfer.